Analysis of the upstream regions governing expression of the chicken cardiac troponin T gene in embryonic cardiac and skeletal muscle cells.

Abstract

The chicken gene encoding cardiac troponin T (cTNT) is expressed in both cardiac and skeletal muscle during early embryonic development, but is specifically repressed in skeletal muscle during fetal development. To determine if the cis-acting sequences governng transcription of a single gene in these two related cell types are the same, we have transfected promoter/upstream segments of the cTNT gene coupled to the bacterial chloramphenicol acetyltransferase gene into primary cultures of early embryonic cardiac and skeletal muscle cells. Using this assay system, chloramphenicol acetyltransferase activity directed by the cTNT promoter/upstream region was between two and three orders of magnitude higher in cardiac or skeletal muscle cells than in fibroblast cells, indicating that cis elements responsible for cell-specific expression reside in this region of the cTNT gene. Deletion experiments showed that a 67-nucleotide DNA segment residing between 268 and 201 nucleotides upstream of the cTNT transcription initiation site is required for cTNT promoter activity in embryonic cardiac cells. This region is not required in embryonic skeletal muscle cells because a cTNT promoter construction containing only 129 upstream nucleotides is transcriptionally active in these cells. These results demonstrate that different cis-acting sequences are required for cTNT expression in early embryonic cardiac and skeletal muscle cells. Nonessential regions residing farther upstream, on the other hand, affected the level of expression of these minimum regions in a similar number in both cell types. The data from these experiemnts indicate, therefore, that transcription of the cTNT promoter in early embryonic cardiac and skeletal muscle cells is governed both by common and divergent regulatory elements in cis and in trans.